One of the major remaining problems in vitamin B12 research is the mechanism of activation via Co-C bond homolysis of 5' deoxyadenosylcobalamin (AdoCb1) by B12-requiring enzymes. Our preliminary studies of complexes of cobalamins with a vitamin B12 binding protein (haptocorrin) from chicken serum suggest that such proteins will serve as excellent models of the interaction of AdoCb1 with enzymes. We thus propose to establish an experimental system for investigating in detail the molecular mechanism by which activation of the coenzyme form of vitamin B12 occurs. In particular, complexes of various cobalamins with the chicken serum haptocorrin will be studied to determine how the binding interaction affects the physical and chemical properties of the cobalamin. The studies will directly address the question of whether such B12 binding proteins can "activate" alkylcobalamins by lowering the energy barrier for thermal carbon-cobalt bond homolysis and will provide a direct experimental approach to a proposed mechanism of enzymic activation of AdoCb1. Measurements to be made include NMR spectroscopic measurements of protein-bound cobalamins to elucidate the effects of the binding interactions on the upper and lower axial ligand bonds of the cobalamins. These will include measurements of the NMR parameters of protein-bound cobalamins enriched in 13C in the imidazole carbon (B2) of the axial nucleotide and of protein-bound cyanocobalt corrins enriched in 15N in the upper cyanide ligand. Measurements of the kinetics of binding of cobalt corrins to the apohaptocorrin will also be made as an adjunct to studies of the kinetics of carbon-cobalt bond dissociation reactions of thermally labile, sterically hindered cobalamins when bound to this B12 binding protein. Such cobalamins will include neopentylcobalamin and benzylcobalamin as well as AdoCb1. The experiments described in this proposal represent the first detailed experimental approach to the question of the molecular mechanism of the enzymatic activation of coenzyme B12. The work represents a major extension of our previous studies on model vitamin B12 systems. Based on our experience and our direct preliminary studies (as described below), we are confident that the proposed experiments, particularly the isotopically labeled NMR studies, will provide valuable molecular insights into the dynamics of the critical cobalamin activation mechanism.